An antibody drug conjugate comprising a phenoxazinyl compound, a pharmaceutical composition and uses thereof

Abstract

This invention relates to an antibody-drug conjugate comprising phenanthroquinollizidine compounds, a pharmaceutical composition thereof, and its uses. The antibody-drug conjugate disclosed in this invention can achieve tumor enrichment, eliminate or reduce the toxic side effects caused by phenanthroquinollizidine compounds acting on non-disease tissues, and improve therapeutic efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antibody-drug conjugate containing phenanthrenequinoloxilidine compounds, a pharmaceutical composition, and its uses. Background Technology

[0002] Phenanthroquinolones are a class of alkaloids mainly found in plants of the genera *Cestrum*, *Cestrum*, *Cestrum*, and some species in the family Asclepiadaceae. These alkaloids possess diverse biological activities, including anti-inflammatory, anticancer, antibacterial, and antifungal properties, with their significant anticancer activity being particularly noteworthy. Screening results for the anticancer activity of these compounds showed that they significantly inhibited 53 selected tumor cell lines, and also exhibited significant inhibitory effects on some multidrug-resistant tumor cells (such as melanoma and lung cancer cells).

[0003] Studies have shown that phenanthroquinolizidine alkaloids involve multiple pathways of action, including inhibiting the synthesis of nucleic acids (including DNA and RNA) in tumor cells, inhibiting protein synthesis, inhibiting the 40S subunit of ribosomes, inhibiting thymidylate synthase, inhibiting dihydrofolate reductase, AP1, AKT / mTOR, AMPK, NF-κB, Cyclin-D1, HIF-1, etc.

[0004] However, no new phenanthrenequinolrizidine alkaloids are currently used clinically. In 1965, Tylocrebrine entered Phase I clinical trials as a treatment for leukemia, but the trials were discontinued due to its severe central nervous system toxicity, which could cause motor incoordination and disorientation in patients. In addition, these compounds also suffer from poor water solubility and unfavorable pharmacokinetic properties (mainly a short half-life).

[0005] Given the unique mechanism of action and the core advantages of these compounds in strongly inhibiting the growth of various tumor cells, as well as their drug-like properties such as high clinical toxicity, poor water solubility, and short half-life, modifying these compounds to address these shortcomings while fully preserving their highly effective anticancer activity has become a key focus and urgent need in current research.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides for the first time an antibody-drug conjugate as shown in Formula I, which exhibits excellent antitumor activity, particularly strong inhibitory activity against the N87 cell line.

[0008] This invention provides an antibody-drug conjugate as shown in Formula I:

[0009] ;

[0010] in,

[0011] Tb is an antibody or its antigen-binding fragment;

[0012] L is a linker that covalently binds Tb and O;

[0013] q is an integer or decimal between 0 and 20, and q is not 0.

[0014] The effects of the invention:

[0015] This invention is the first to apply phenanthroline-quinolrilidine compounds to antibody-drug conjugates (ADCs), resulting in ADC molecules with these compounds as the payload. This approach enhances the efficacy by enriching the payload within tumor tissues / cells and reducing drug distribution in non-target tissues, thus decreasing toxicity and further improving therapeutic effects. Furthermore, this ADC molecule exhibits a faster payload release rate, enabling its use in the treatment of various hematologic malignancies and solid tumors, and demonstrates better therapeutic efficacy than its counterparts in the phenanthroline-quinolrilidine class. Moreover, this ADC molecule may show unexpected benefits in overcoming drug resistance, giving it greater clinical application value. Detailed Implementation

[0016] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0017] Terminology Explanation:

[0018] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0019] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition) for details.

[0020] The term "pharmaceutical excipients" refers to all substances contained in a pharmaceutical preparation other than the active pharmaceutical ingredient, and are generally divided into two main categories: excipients and additives. For details, please refer to the Pharmacopoeia of the People's Republic of China (2020 Edition) and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).

[0021] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0022] The term "treatment" refers to eliminating the cause of an illness or relieving symptoms.

[0023] The term "prevention" refers to reducing the risk of developing a disease.

[0024] The term "patient" refers to any animal, typically a mammal such as a human, that requires treatment or prevention of disease. Mammals include, but are not limited to: cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans.

[0025] The term "therapeutic effective dose" refers to the amount given to a patient that is sufficient to effectively treat the disease. Therapeutic effective doses will vary depending on the type of compound, the type of disease, the severity of the disease, the patient's age, etc., but may be adjusted as appropriate by those skilled in the art.

[0026] The term "antibody-drug conjugate" refers to a substance obtained by linking a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present invention, the bioactive compound fragment and the target moiety are linked by a linker. The linker is cleavable under specific conditions (e.g., intracellular low pH) or specific actions (e.g., lysosomal proteases), thereby separating the bioactive compound fragment from the target moiety or the antibody or its antigen-binding fragment. In some embodiments of the present invention, the linker comprises cleavable or non-cleavable units, such as peptides or disulfide bonds. In some embodiments of the present invention, the bioactive compound fragment and the target moiety or the antibody or its antigen-binding fragment are directly linked by a covalent bond, which is cleavable under specific conditions or actions, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0027] The term "linker" or "linker" refers to a segment that links a fragment of a bioactive compound (drug molecule) to an antibody portion.

[0028] The term "methyl sulfone" refers to "-SO2-CH3".

[0029] In this invention, regarding "the 1-position of L is connected to Tb via an S atom," those skilled in the art will understand that the 1-position of L is connected to the thiol group inherent in Tb (such as an antibody) after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing it with the reducing agent TCEP to generate a thiol group -SH). In other words, the -S- between L and Tb is not an additional external sulfur atom. For example, the -S- is not an additional external sulfur atom, but rather a -S- formed by connecting the 1-position of the thiol group inherent in Tb itself after the disulfide bond is opened.

[0030] The term "antibody" is used in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this invention, "antibody" and "immunoglobulin" are used interchangeably.

[0031] The term "monoclonal antibody" refers to an antibody derived from a largely homogeneous group of antibodies, meaning that the antibodies constituting this cluster are identical except for a small number of possible natural mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, contain different antibodies targeting different determinants (epitopes). Besides specificity, a key advantage of monoclonal antibodies is that their synthesis is unaffected by contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a largely homogeneous group of antibodies, and should not be interpreted as requiring a special method of preparation.

[0032] In some embodiments of the invention, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to one, a class, or a subclass of antibody, while the remainder is identical or homologous to another, a different class, or a different subclass of antibody, provided they possess the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present invention include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0033] The term "antibody fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region fragments, diantibodies, linear antibodies, and single-chain antibody molecules.

[0034] The term "bispecific antibody," also known as "bifunctional antibody-drug conjugate," refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a conjugate arm. This conjugate retains the activity of each antibody and thus has both bifunctionality and bispecificity.

[0035] The term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies. The former is an antibody that has the binding specificity of three different antigens, while the latter is an antibody that has the binding specificity of four different antigens.

[0036] The term "intact antibody" refers to an antibody that contains an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be natural sequences (e.g., human natural constant region sequences) or amino acid sequence variants thereof. Intact antibodies are preferably intact antibodies with one or more effector functions.

[0037] The term "probody" is a modified antibody, including an antibody or antibody fragment that specifically binds to its target and can be coupled with a masking group, wherein the masking group refers to a cleavage constant that is at least 100 times, 1000 times, or 10000 times greater than the cleavage constant that is not coupled to the target of an antibody or antibody fragment.

[0038] In this invention, the “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which hypervariable region residues of a human recipient immunoglobulin have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibodies) possessing the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with non-human residues. Furthermore, humanized antibodies may also contain residues not present in the recipient or donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies generally contain at least one, typically two, variable regions, where all or almost all hypervariable loops correspond to non-human immunoglobulins, while the FRs are entirely or almost entirely human immunoglobulin sequences. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc, typically human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-329; and Presta, 1992, Curr Op Struct Bwl 2: 593-596.

[0039] Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant regions. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different antibody classes are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known in the art.

[0040] In this invention, although the amino acids in the antibodies are mostly L-amino acids, this is not a limitation. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less prone to degradation in the oral cavity, intestines, or plasma than peptides containing only L-amino acids.

[0041] The monoclonal antibodies used in this invention can be produced by many methods. For example, the monoclonal antibodies used in this invention can be obtained by hybridoma methods using cells from many species, including mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256: 495), or by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352: 624-628; and Marks et al., 1991, Journal of Molecular Biology, 222: 581-597).

[0042] In some embodiments of the present invention, Tb is trastuzumab or pertuzumab. Trastuzumab is a monoclonal antibody against Her2, the amino acid sequence of which is known to those skilled in the art, and an illustrative sequence can be found, for example, in CN103319599. Exemplary heavy and light chain sequences of pertuzumab can be found in SEQ ID No. 16 and SEQ ID No. 15 of US7560111. The terminal Lys position is readily deleted but does not affect biological activity; see Dick, LW et al., Biotechnol. Bioeng., 100: 1132-1143.

[0043] In this invention, ErbB2 and Her2 / neu are used interchangeably, both representing the native sequence of the human Her2 protein (Genebank accession number: X03363, see, for example, Semba et al., 1985, PNAS, 82: 6497-6501; and Yamamoto et al., 1986, Nature, 319: 230-234) and its functional derivatives, such as amino acid sequence variants. ErbB2 represents the gene encoding human Her2, and neu represents the gene encoding rat p185neu. In some embodiments, the compounds or conjugates of this invention are capable of inhibiting or killing cells expressing the ErbB2 receptor, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, kidney cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells, or liver cancer cells.

[0044] In a first aspect, the present invention provides an antibody-drug conjugate as shown in Formula I:

[0045] ;

[0046] in,

[0047] Tb is an antibody or its antigen-binding fragment;

[0048] L is a linker that covalently binds Tb and O;

[0049] q is an integer or decimal between 0 and 20, and q is not 0.

[0050] In some embodiments, the Tb is an anti-Her2 antibody or its antigen-binding fragment.

[0051] In some implementations, Tb is trastuzumab.

[0052] In some implementations, L is any of the following structures:

[0053] , , , ,

[0054] In this configuration, position 1 is connected to Tb via an S atom, and position 2 is connected to O.

[0055] In some implementations, q is any integer from 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

[0056] In some implementations, q is 6.

[0057] In some implementations, q is 7.

[0058] In some implementations, q is 8.

[0059] Secondly, the present invention provides an antibody-drug conjugate having any of the following structures:

[0060] , , , ,

[0061] Where q is 8.

[0062] Thirdly, the present invention provides a drug linker conjugate as shown in Formula II:

[0063] ;

[0064] in,

[0065] Lg is methyl sulfone group;

[0066] L is a linker that covalently binds Lg and O.

[0067] In some implementations, L is any of the following structures:

[0068] , , , ,

[0069] In this configuration, bit 1 is connected to Lg, and bit 2 is connected to O.

[0070] Fourthly, the present invention provides a drug linker conjugate, characterized in that the drug linker conjugate has any of the following structures:

[0071] , , , .

[0072] Fifthly, the present invention provides a compound as shown in Formula III or a pharmaceutically acceptable salt thereof:

[0073] .

[0074] In a sixth aspect, the present invention provides a compound as shown in Formula III-A or a pharmaceutically acceptable salt thereof:

[0075] .

[0076] In a seventh aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as shown in Formula I, and one or more pharmaceutical excipients;

[0077] ;

[0078] in,

[0079] Tb is an antibody or its antigen-binding fragment;

[0080] L is a linker that covalently binds Tb and O;

[0081] q is an integer or decimal between 0 and 20, and q is not 0.

[0082] In some embodiments, the Tb is an anti-Her2 antibody or its antigen-binding fragment.

[0083] In some implementations, Tb is trastuzumab.

[0084] In some implementations, L is any of the following structures:

[0085] , , , ,

[0086] In this configuration, position 1 is connected to Tb via an S atom, and position 2 is connected to O.

[0087] In some implementations, q is any integer from 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

[0088] In some implementations, q is 6.

[0089] In some implementations, q is 7.

[0090] In some implementations, q is 8.

[0091] Eighthly, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate with any of the following structures, and one or more pharmaceutical excipients;

[0092] , , , ,

[0093] Where q is 8.

[0094] In a ninth aspect, the present invention provides the use of the antibody-drug conjugates or pharmaceutical compositions described above in the preparation of medicaments for treating and / or preventing diseases related to abnormal cell activity.

[0095] In some implementations, the disease associated with abnormal cell activity is selected from cancer.

[0096] In some implementations, the cancer is selected from solid tumors or hematologic malignancies.

[0097] In some implementations, the cancer is selected from cancers associated with Her2.

[0098] In some implementations, the cancer is selected from breast cancer, ovarian cancer, stomach cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer, bladder cancer, and liver cancer.

[0099] In some implementations, the cancer is selected from breast cancer, stomach cancer, and lung cancer.

[0100] In some implementations, the cancer is selected from gastric cancer.

[0101] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features, and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements. Implementation conditions not specified are generally those in routine experiments or selected according to the product instructions. The reagents and raw materials used in the present invention are all commercially available.

[0102] The abbreviations used in this invention embodiment have the meanings shown in Table 1:

[0103] Table 1

[0104]

[0105] The structures of the compounds described in the embodiments of this invention were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0106] The following examples 1 The 1H NMR was performed using a Bruker 400 MHz NMR spectrometer, and the chemical shift δ values ​​were expressed in ppm. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); the internal standard was tetramethylsilane (TMS).

[0107] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the following examples are shown below:

[0108] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated.

[0109] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0110] DAR value determination method:

[0111] Sample preparation: Take 50 μg of ADC sample, dilute it with ultrapure water to 0.5 mg / mL, then add DTT (1 μL, 1 M), mix well, centrifuge and take the supernatant for injection.

[0112] The instruments, liquid chromatography parameters, and mass spectrometry parameters used in this method are shown in Tables 2, 3, and 4.

[0113] Table 2

[0114]

[0115] Table 3

[0116]

[0117] Table 4

[0118]

[0119] Example 1 Synthesis of intermediates

[0120] Example 1.1: Synthesis of INT1

[0121]

[0122] Step 1:

[0123] Compound INT1-1 (10 g, 50.5 mmol) was dissolved in tetrahydrofuran (100 mL), and INT1-2 (10.35 g, 50.5 mmol), bis(triphenylphosphine)palladium dichloride (3.5 g, 5.05 mmol), cuprous iodide (1.92 g, 10.1 mmol), and triethylamine (15.3 g, 151.5 mmol) were added. The mixture was stirred overnight at 70 °C under a nitrogen atmosphere. The reaction solution was cooled, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain the target compound INT1-3 (5.2 g).

[0124] LCMS (ESI) [M+H] + = 323.0;

[0125] 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 2H), 3.98 (s, 2H), 3.57 (t, J =6.2 Hz, 2H), 2.57 – 2.54 (m, 2H), 2.52 (s, 3H), 1.83 – 1.76 (m, 2H), 1.42 (s,9H).

[0126] Step Two:

[0127] Compound INT1-3 (1.0 g, 3.1 mmol) was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (3.54 g, 31 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was then concentrated under reduced pressure to give compound INT1-4 (1.5 g).

[0128] LCMS (ESI) [M+H] + = 267.0;

[0129] 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 2H), 4.03 (s, 2H), 3.59 (t, J =6.2 Hz, 2H), 2.58 – 2.53 (m, 2H), 2.53 (s, 3H), 1.84 – 1.77 (m, 2H).

[0130] Step 3:

[0131] Compound INT1-4 (1.5 g, 5.6 mmol) was dissolved in tetrahydrofuran (20 mL) and water (20 mL), and Oxone (10.3 g, 16.8 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran. The aqueous phase was extracted three times with ethyl acetate (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by reversed-phase chromatography (acetonitrile:water (containing 0.05% HCl) = 5%-50%) to obtain the target compound INT1 (0.4 g).

[0132] LCMS (ESI) [M+H] + = 299.1;

[0133] 1 H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.11 (s, 2H), 4.03 (s, 2H), 3.60 (t, J = 6.2 Hz, 2H), 3.41 (s, 3H), 2.63 (t, J = 7.1 Hz, 2H), 1.87 – 1.80(m, 2H).

[0134] Example 1.2: Synthesis of INT2

[0135]

[0136] Step 1:

[0137] Compound INT2-1 (5 g, 17.84 mmol) was added to trifluoroethanol (30 mL) under argon protection and stirred at 40 °C to dissolve. Then, DMF hydrogen chloride solution (2.1 mL, 0.4 mol / L) was added and the reaction was stirred at 40 °C for 2.5 h. After the reaction solution cooled to room temperature, it was added to an aqueous sodium bicarbonate solution (120 mL, 1%, W / V), stirred, filtered, and the filter cake was dried to obtain the target compound INT2-2 (5.3 g).

[0138] LCMS (ESI) [M+Na] + = 343.2.

[0139] 1 H NMR (400 MHz, DMSO-d6) δ 8.88 (t, J = 6.5 Hz, 1H), 7.54 (t, J = 6.0Hz, 1H), 7.40-7.32 (m, 5H), 5.07 (s, 2H), 4.70 (d, J = 6.8 Hz, 2H), 4.03 (q,J = 9.4 Hz, 2H), 3.68 (d, J = 6.1 Hz, 2H).

[0140] Step Two:

[0141] Compound INT2-2 (1.00 g, 3.29 mmol) was dissolved in THF (5 mL), and Pd / C (5%, 50 mg) was added under nitrogen protection. After hydrogen purging, the reaction was carried out at room temperature for 3 h. DMF (5 mL) was added to the reaction solution, filtered, and concentrated to remove THF, yielding a DMF solution of the target compound INT2-3, which was directly used in the next step of the reaction.

[0142] LCMS (ESI) [M+H] + = 187.21.

[0143] The 1H NMR spectrum of the HOBt salt of compound INT2-3 is as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.18(brs, 1H), 7.79-7.71 (m, 1H), 7.49-7.41 (m, 1H), 7.25-7.18 (m, 2H), 4.74 (d,J = 4.9 Hz, 2H), 4.06 (q, J = 9.4 Hz, 2H), 3.47 (s, 2H).

[0144] Step 3:

[0145] Compounds INT2-4 (750 mg, 1.81 mmol) and INT2-3 (337 mg, 1.81 mmol) were dissolved in DMF (5 mL), cooled to 0 °C, and then DMTMM (480 mg, 1.99 mmol) was added. The reaction was continued for 1 h, and the reaction was monitored by LCMS. The reaction solution was diluted with DCM (30 mL), and after adding saturated NaHCO3 solution (30 mL), the mixture was stirred for 5 min. The mixture was separated, and the aqueous phase was extracted once again with DCM (20 mL). The organic phases were combined, dried, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (MeOH:DCM=0~10%) to obtain the target compound INT2-5 (950 mg).

[0146] LCMS (ESI) [M+Na] + =604.3.

[0147] 1 H NMR (400 MHz, DMSO-d6) δ 8.72 (t, J = 6.7 Hz, 1H), 8.36 (t, J = 5.6Hz, 1H), 8.17 (d, J = 7.9 Hz, 1H), 8.03 (t, J = 5.4 Hz, 1H), 7.51 (t, J = 5.8Hz, 1H), 7.42 – 7.30 (m, 5H), 7.29 – 7.19 (m, 5H), 5.06 (s, 2H), 4.71 (d, J =6.9 Hz, 2H), 4.57 – 4.51 (m, 1H), 4.03 (q, J = 9.4 Hz, 2H), 3.84 – 3.70 (m,3H), 3.67 – 3.61 (m, 3H), 3.09 (dd, J = 13.8, 4.5 Hz, 1H), 2.83 (dd, J =13.7, 9.7 Hz, 1H).

[0148] Step Four:

[0149] Compound INT2-5 (800 mg, 1.38 mmol) was dissolved in methanol (10 mL), and palladium on carbon (73 mg, wt 10%) was added under nitrogen protection. The mixture was purged with hydrogen three times, and the reaction solution was stirred under hydrogen atmosphere for 16 h. The reaction was monitored by LCMS, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT2-6 (590 mg).

[0150] LCMS (ESI) [M+H] + =448.29.

[0151] 1 H NMR (400 MHz, DMSO-d6) δ 8.74 (t, J = 6.7 Hz, 1H), 8.40 (t, J = 5.7Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.33 – 7.18 (m, 5H), 4.71(d, J = 6.8 Hz, 2H), 4.54 (td, J = 9.3, 4.6 Hz, 1H), 4.04 (q, J = 9.4 Hz, 2H), 3.85 – 3.61 (m, 4H), 3.21 – 3.05 (m, 3H), 2.83 (dd, J = 13.8, 9.7 Hz, 1H).

[0152] Step 5:

[0153] Compounds INT2-6 (501 mg, 1.12 mmol) and INT2-7 (300 mg, 1.12 mmol) were dissolved in DMF (5 mL), cooled to 0 °C, and then DMTMM (341 mg, 1.23 mmol) was added. The mixture was stirred for 1 h. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.05% TFA = 5%-50%) to obtain the target compound INT2-8 (610 mg).

[0154] LCMS (ESI) [M+H] + =698.30.

[0155] 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 8.79 – 8.72 (m, 1H), 8.38(dd, J = 13.1, 7.4 Hz, 1H), 8.23 ​​(t, J = 5.7 Hz, 1H), 8.18 (d, J = 8.0 Hz,1H), 8.10 (t, J = 5.6 Hz, 1H), 7.31 – 7.17 (m, 5H), 4.75 – 4.66 (m, 2H), 4.52(tt, J = 11.8, 5.8 Hz, 1H), 4.04 (dd, J = 18.7, 9.3 Hz, 3H), 3.88 – 3.70 (m,4H), 3.63 (dd, J = 16.8, 5.3 Hz, 1H), 3.43 (s, 3H), 3.09 (dd, J = 13.8, 4.4Hz, 1H), 2.84 (dd, J = 13.8, 9.7 Hz, 1H), 2.60 (t, J = 7.1 Hz, 2H), 2.36 (t,J = 7.3 Hz, 2H), 1.91 – 1.77 (m, 2H).

[0156] Step Six:

[0157] Compound INT2-8 (100 mg, 0.14 mmol) and glycolic acid (53 mg, 0.70 mmol) were dissolved in DMF (1 mL), and then HCl / DMF (4 N, 0.1 mL) was added. The mixture was reacted at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 5%-50%) to obtain the target compound INT2 (50 mg).

[0158] LCMS (ESI) [MH] + =672.35.

[0159] 1 H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 2H), 8.70–8.64 (m, 1H), 8.38 –8.07 (m, 4H), 7.29 – 7.21 (m, 5H), 4.66 (d, J = 6.5 Hz, 2H), 4.57 – 4.48 (m,1H), 3.98 – 3.88 (m, 2H), 3.80 – 3.73 (m, 4H), 3.68 –3.64 (m, 2H), 3.44 (s,3H), 3.10 (dd, J = 13.8, 4.7 Hz, 1H), 2.85 (dd, J = 13.9, 9.6 Hz, 1H), 2.61(t, J = 7.1 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 1.86 (p, J = 7.2 Hz, 2H).

[0160] Example 1.3: Synthesis of INT3

[0161]

[0162] Step 1:

[0163] INT3-1 (20 g, 140.8 mmol) was dissolved in THF (400 mL), stirred in an ice bath for 15 min, and then triphenylphosphine (36.9 g, 140.8 mmol) was added. Then imidazole (9.574 g, 140.8 mmol) and iodine (35.763 g, 140.8 mmol) were added sequentially. The mixture was brought back to room temperature and stirred for 2 h. The reaction was quenched with saturated sodium bisulfite (50 mL), and then extracted with methyl tert-butyl ether (500 mL x 3). The organic phases were combined, dried, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether) to obtain the target compound INT3-2 (28.8 g).

[0164] 1 H NMR (400 MHz, CDCl3) δ 3.21 (t, J = 7.5 Hz, 2H), 2.78 (t, J = 7.5Hz, 2H), 0.16 (s, 9H).

[0165] Step Two:

[0166] Diisopropylamine (2.22 g, 21.78 mmol) was dissolved in THF (15 mL), cooled to -78 °C, and n-butyllithium (16 mL, 1.6 N) was added dropwise. After the addition was complete, the mixture was stirred at this temperature for 30 min. Methyl isobutyrate (2.22 g, 21.78 mmol) was then added, and the mixture was heated to 0 °C and stirred for 30 min. The mixture was then cooled again to -78 °C, and a tetrahydrofuran (2 mL) solution of compound INT3-3 (5 g, 19.8 mmol) was added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 h. The mixture was then cooled to 0 °C and quenched with saturated ammonium chloride (10 mL). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by column chromatography (petroleum ether) to obtain the target compound INT3-4 (3.2 g).

[0167] LCMS (ESI) [M+H] + = 227.3.

[0168] 1 H NMR (400 MHz, CDCl3) δ 3.66 (s, 3H), 2.23 – 2.11 (m, 2H), 1.85 –1.75 (m, 2H), 1.17 (s, 6H), 0.17 – 0.09 (m, 9H).

[0169] Step 3:

[0170] Compounds INT3-4 (5 g, 22.12 mmol) and INT3-5 (4.512 g, 22.12 mmol) were dissolved in acetonitrile (10 mL) and THF (30 mL). Tetra(triphenylphosphine)palladium (2.554 g, 2.212 mmol), cuprous iodide (840 mg, 4.424 mmol), cesium fluoride (6.724 g, 44.24 mmol), and triethylamine (8.936 g, 88.48 mmol) were added sequentially. The mixture was heated to 80 °C and stirred for 16 h under an argon atmosphere. The mixture was then diluted with ethyl acetate (150 mL), filtered, concentrated, and the crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target compound INT3-6 (3.1 g).

[0171] LCMS (ESI) [M+H] + = 279.2.

[0172] 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 2H), 3.66 (s, 3H), 2.55 (s, 3H), 2.39 (dd, J = 8.8, 7.3 Hz, 2H), 1.89 (dd, J = 8.8, 7.2 Hz, 2H), 1.22 (s, 6H).

[0173] Step Four:

[0174] Compound INT3-6 (3.1 g, 11.15 mmol) was dissolved in water (44 mL), methanol (22 mL), and tetrahydrofuran (44 mL). Sodium hydroxide (1.338 g, 33.45 mmol) was added, and the mixture was stirred at room temperature for 16 h. The pH was adjusted to 2 with 1N hydrochloric acid, the mixture was concentrated under reduced pressure, filtered, and the filter cake was purified by column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain the target compound INT3-7 (2.5 g).

[0175] LCMS (ESI) [M+H] + = 265.2.

[0176] 1 H NMR (400 MHz, CDCl3) δ 8.48 (s, 2H), 2.55 (s, 3H), 2.45 (d, J = 8.2Hz, 2H), 1.96 – 1.89 (m, 2H), 1.25 (s, 6H).

[0177] Step 5:

[0178] Compound INT3-7 (3.5 g, 13.25 mmol) was dissolved in tetrahydrofuran (30 mL) and water (30 mL), and Oxone (20.35 g, 33.14 mmol) was added. The mixture was stirred at room temperature for 70 min. The reaction solution was extracted with ethyl acetate (200 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.01% HCl = 10%-90%) to obtain the target compound INT3 (3.4 g).

[0179] LCMS (ESI) [M+H] + = 297.2.

[0180] 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 2H), 3.34 (s, 3H), 2.53 (t, J = 7.7Hz, 2H), 1.95 (t, J = 7.8 Hz, 2H), 1.27 (s, 6H).

[0181] Example 1.4: Synthesis of INT4

[0182]

[0183] Step 1:

[0184] Weigh INT4-1 (2.0 g, 9.0 mmol) into a 100 mL round-bottom flask, and add a 30 mL solution of potassium carbonate (124 mg, 0.9 mmol) in water and an aqueous solution of formaldehyde (1.31 g, 16.2 mmol, 37%) sequentially. Stir the reaction mixture at room temperature for 4 h. Filter the reaction mixture, wash the filter cake once with water (5 mL), and dry the resulting solid to obtain the target compound INT4-2 (500 mg).

[0185] LCMS (ESI) [M+Na] + =275.26.

[0186] 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (t, J = 6.0 Hz, 1H), 7.36 (d, J = 5.4Hz, 6H), 5.53 (t, J = 6.0 Hz, 1H), 5.07–4.94 (m, 2H), 4.60–4.37 (m, 2H), 4.01(p, J = 7.3 Hz, 1H), 1.20 (d, J = 7.1 Hz, 3H).

[0187] Step Two:

[0188] INT4-2 (500 mg, 1.98 mmol) was dissolved in trifluoroethanol (3 mL), and a DMF solution of hydrochloric acid (0.25 mL, 0.1 mmol, 0.4 M) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then added dropwise to a 2% sodium bicarbonate solution (12 mL), and stirred for 2 h. A solid precipitated out. The solid was filtered, and the filter cake was dried to obtain the target compound INT4-3 (550 mg).

[0189] LCMS (ESI) [M+Na] + =357.19.

[0190] 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (t, J = 6.4 Hz, 1H), 7.53 (d, J = 7.4Hz, 1H), 7.39–7.27 (m, 5H), 5.08–4.96 (m, 2H), 4.70–4.61 (m, 2H), 4.07–3.93(m, 3H), 1.22 (d, J = 7.2 Hz, 3H).

[0191] Step 3:

[0192] Compound INT4-3 (450 mg, 1.35 mmol) was dissolved in methanol (9 mL), and palladium on carbon (45 mg, 10%) was added under nitrogen protection. The mixture was purged three times with hydrogen, and the reaction solution was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT4-4 (250 mg).

[0193] LCMS (ESI) [M+H] + =201.18.

[0194] 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 4.67 (s, 2H), 4.02 (q, J =9.4 Hz, 2H), 3.29 (q, J = 6.9 Hz, 1H), 1.83 (s, 2H), 1.13 (d, J = 6.9 Hz,3H).

[0195] Step Four:

[0196] INT4-4 (250 mg, 1.25 mmol) and INT4-5 (570 mg, 1.5 mmol) were dissolved in DMF (3 mL), cooled to 0 °C, and DMTMM (420 mg, 1.5 mmol) was added. After the addition was complete, the reaction solution was brought back to room temperature and stirred for 2 h. The reaction solution was purified by direct reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound INT4-6 (350 mg).

[0197] LCMS (ESI) [M+Na] + =587.28.

[0198] 1 H NMR (400 MHz, DMSO-d6) δ 8.78 (t, J = 6.9 Hz, 1H), 8.00 (d, J = 7.3Hz, 2H), 7.89 (d, J = 7.4 Hz, 2H), 7.71 (t, J = 7.0 Hz, 2H), 7.52 (d, J = 7.3Hz, 1H), 7.42 (t, J = 7.1 Hz, 2H), 7.37–7.29 (m, 2H), 4.73–4.60 (m, 2H), 4.32–4.14 (m, 5H), 4.05 (t, J = 7.3 Hz, 1H), 3.98 (q, J = 9.4 Hz, 2H), 1.21(dd, J = 7.2, 4.6 Hz, 9H).

[0199] Step 5:

[0200] Compound INT4-6 (100 mg, 0.18 mmol) was dissolved in DMF (1.5 mL), and diethylamine (0.15 mL) was added. The reaction mixture was stirred at room temperature for 2 h. 5 mL of tetrahydrofuran was added to the reaction mixture, and the solution was concentrated under reduced pressure. The above operation was repeated three times to obtain a DMF solution of the target compound INT4-7.

[0201] LCMS (ESI) [M+H] + =343.21

[0202] Step Six:

[0203] Add INT2-7 (58 mg, 0.22 mmol) to the DMF solution of INT4-7 obtained in step 5, cool to 0 °C, and add DMTMM (60 mg, 0.22 mmol). After the addition is complete, stir the reaction solution at room temperature for 2 h. Purify the reaction solution by direct reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound INT4-8 (96 mg).

[0204] LCMS (ESI) [M+Na] + =615.36.

[0205] 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 2H), 7.95 (t, J = 6.9 Hz, 1H), 7.27 (d, J = 7.1 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.15 (d, J = 7.0 Hz, 1H),3.90–3.78 (m, 2H), 3.47–3.33 (m, 3H), 3.21–3.10 (m, 2H), 2.58 (s, 3H), 1.73(t, J = 7.1 Hz, 2H), 1.48 (t, J = 7.7 Hz, 2H), 1.04–0.91 (m, 2H), 0.44–0.34 (m, 9H).

[0206] Step Seven:

[0207] Compound INT4-8 (100 mg, 0.17 mmol) and glycolic acid (65 mg, 0.85 mmol) were dissolved in DMF (1.7 mL, 1.5 M) solution in hydrochloric acid, and the reaction solution was stirred at 30 °C for 2 h. The reaction solution was then purified directly by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 5%-50%) to obtain the target compound INT4 (20 mg).

[0208] LCMS (ESI) [M+Na] + =591.28.

[0209] Example 1.5: Synthesis of INT5

[0210]

[0211] Step 1:

[0212] Compounds INT2-6 (313.2 mg, 0.70 mmol) and INT1 (210.0 mg, 0.70 mmol) were dissolved in DMF (5 mL), cooled to 0 °C, and then DMTMM (213.0 mg, 0.77 mmol) was added. The mixture was stirred at 0 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reverse-phase column chromatography (acetonitrile:H2O containing 0.05% FA = 5%-50%) to obtain the target compound INT5-1 (390 mg).

[0213] LCMS (ESI) [M+Na]+ =750.47.

[0214] 1 H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 2H), 8.74 (t, J = 6.8 Hz, 1H), 8.38 (t, J = 5.7 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.12 (t, J = 5.6 Hz, 1H),7.97 (t, J = 5.7 Hz, 1H), 7.33 – 7.18 (m, 5H), 4.71 (d, J = 6.9 Hz, 2H),4.57– 4.50 (m, 1H), 4.04 (dd, J = 18.7, 9.3 Hz, 2H), 3.96 (s, 2H), 3.84 –3.70 (m, 5H), 3.68 – 3.60 (m, 3H), 3.44 (s, 3H), 3.08 (dd, J = 13.8, 4.4 Hz, 1H), 2.83 (dd, J = 13.7, 9.7 Hz, 1H), 2.70 (t, J = 7.1 Hz, 2H), 1.95– 1.85(m,2H).

[0215] Step Two:

[0216] Compound INT5-1 (150.0 mg, 0.21 mmol) and glycolic acid (79.9 mg, 1.05 mmol) were dissolved in DMF (1 mL), followed by the addition of HCl / DMF (0.16 mL, 4N). The mixture was stirred at room temperature for 4 h after the addition was complete. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound INT5 (65 mg).

[0217] LCMS (ESI) [MH] - =702.40.

[0218] 1H NMR (400 MHz, DMSO-d6) 12.63 (s, 1H), 9.14 (s, 2H), 8.59 (t, J =6.7 Hz, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 8.12 (t, J =5.6 Hz, 1H), 7.97 (t, J = 5.7 Hz, 1H), 7.31 – 7.18 (m, 5H), 4.64 (d, J = 6.7Hz, 2H), 4.57– 4.50 (m, 1H), 4.01 (s, 2H), 3.96 (s, 2H), 3.82 – 3.68 (m, 5H), 3.68 – 3.60 (m, 3H), 3.44 (s, 3H), 3.09 (dd, J = 13.8, 4.4 Hz, 1H), 2.83 (dd,J = 13.8, 9.8 Hz, 1H), 2.70 (t, J = 7.1 Hz, 2H), 1.95– 1.85(m, 2H).

[0219] Example 1.6: Synthesis of INT6

[0220]

[0221] Step 1:

[0222] INT3 (207 mg, 0.7 mmol) and DMTMM (213 mg, 0.77 mmol) were added sequentially to a DMF solution (5 mL) of compound INT2-6 (315 mg, 0.7 mmol). After addition, the mixture was brought to room temperature and stirred for 2 h. The reaction was monitored by LCMS. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound INT6-1 (270 mg).

[0223] LCMS (ESI) [M+Na] + =748.48.

[0224] 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 2H), 8.71 (t, J = 6.8 Hz, 1H), 8.35 (t, J = 5.8 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H),7.89 (t, J = 5.6 Hz, 1H), 7.29 – 7.20 (m, 4H), 7.20 – 7.13 (m, 1H), 4.68 (d,J = 7.4 Hz, 2H), 4.56 – 4.46 (m, 1H), 4.06 – 3.95 (m, 2H), 3.81 – 3.57 (m, 6H), 3.40 (s, 3H), 3.10 – 3.01 (m, 1H), 2.86 – 2.76 (m, 1H), 2.55 – 2.51 (m,2H), 1.83 (t, J = 8.5 Hz, 2H), 1.14 (s, 6H).

[0225] Step Two:

[0226] Compound INT6-1 (270 mg, 0.37 mmol) and glycolic acid (140 mg, 1.85 mmol) were dissolved in hydrochloric acid / DMF (5.6 mL, 0.1 M), and the reaction mixture was stirred at 30 °C for 2 h. The reaction was monitored by LCMS. The reaction mixture was directly purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.1% FA = 10%-90%) to obtain the target compound INT6 (130 mg).

[0227] LCMS (ESI) [M+Na] + =724.48.

[0228] 1H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 9.07 (s, 2H), 8.55 (t, J =6.6 Hz, 1H), 8.31 (t, J = 6.0 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.98 (t, J =5.6 Hz, 1H), 7.89 (t, J = 5.7 Hz, 1H), 7.24 (d, J = 5.8 Hz, 4H), 7.17 (td, J= 6.0, 2.4 Hz, 1H), 4.61 (d, J = 6.8 Hz, 2H), 4.56 – 4.46 (m, 1H), 3.98 (s, 2H), 3.81 – 3.55 (m, 6H), 3.41 (s, 3H), 3.11 – 3.02 (m, 1H), 2.86 – 2.76 (m,1H), 2.55 – 2.50 (m, 2H), 1.88 – 1.79 (m, 2H), 1.14 (s, 6H).

[0229] Example 2 Synthesis of toxin molecules (phenanthroquinolrizidine compounds)

[0230] Example 2.1: Synthesis of D-2

[0231]

[0232] Step 1:

[0233] Compound D-1 (30 mg, 0.083 mmol) was dissolved in N,N-dimethylformamide (2 mL), and glycolic acid (63 mg, 0.828 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (62 mg, 0.166 mmol) and N,N-diisopropylethylamine (27 mg, 0.207 mmol) were added. The reaction solution was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile:H2O containing 0.05% trifluoroacetic acid = 5%-50%) to obtain trifluoroacetate of target compound D-2 (8.29 mg).

[0234] LCMS (ESI) [M+H] + = 421.4.

[0235] 1H NMR (400 MHz, DMSO-d6) δ 10.23 (s, 1H), 9.96 (s, 1H), 9.00 (s, 1H), 8.22 (d, J = 8.6 Hz, 1H), 8.03 (s, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.37 (s,1H), 5.93 (s, 1H), 5.07 (d, J = 16.2 Hz, 1H), 4.57 (d, J = 15.9 Hz, 1H), 4.09(s, 2H), 4.03 (s, 3H), 3.98 (s, 3H), 3.79 (d, J = 10.6 Hz, 1H), 3.59 (d, J =14.1 Hz, 2H), 3.23 – 3.08 (m, 2H), 2.33 – 2.23 (m, 1H), 2.01 – 1.88 (m, 2H), 1.80 – 1.55 (m, 3H).

[0236] Example 3 Synthesis of drug linker conjugates

[0237] Example 3.1: Synthesis of DL-1

[0238]

[0239] Step 1:

[0240] Compounds INT2 (20 mg, 0.03 mmol) and D-1-1 (11 mg, 0.03 mmol, synthetic method see J.Med. Chem. 2015, 58, 7749−7762) were dissolved in DMF (1.5 mL), cooled to 0 °C, and DMTMM (7.8 mg, 0.033 mmol) was added. After the addition was complete, the mixture was allowed to return to room temperature and stirred for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 5%–50%) to obtain the target compound DL-1 (17.9 mg).

[0241] LCMS (ESI) [M+H] + =1017.8.

[0242] 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.09 (s, 2H), 8.98 (d, J =2.4 Hz, 1H), 8.78 (t, J = 6.3 Hz, 1H), 8.40 (t, J = 5.8 Hz, 1H), 8.24 – 8.16(m, 2H), 8.10 (t, J = 5.7 Hz, 1H), 8.03 – 7.96 (m, 2H), 7.85 (d, J = 9.1 Hz,1H), 7.34 (s, 1H), 7.31 – 7.17 (m, 5H), 4.77 (d, J = 6.7 Hz, 2H), 4.57 – 4.49(m, 1H), 4.38 (d, J = 16.0 Hz, 1H), 4.17 (s, 2H), 4.02 (s, 3H), 3.98 (s, 3H),3.84 – 3.76 (m, 2H), 3.74 (d, J = 5.8 Hz, 2H), 3.68 – 3.61 (m, 2H), 3.42 (s,3H), 3.24 – 3.17 (m, 2H), 3.15 – 3.07 (m, 2H), 2.89 – 2.77 (m, 2H), 2.60 –2.55 (m, 2H), 2.37 – 2.33 (m, 2H), 2.29 – 2.18 (m, 1H), 2.07 – 1.98 (m, 2H), 1.90 – 1.58 (m, 5H), 1.50 – 1.38 (m, 2H).

[0243] Example 3.2: Synthesis of DL-2

[0244]

[0245] Step 1:

[0246] Compounds INT4 (16 mg, 0.028 mmol) and D-1-1 (10 mg, 0.028 mmol) were dissolved in DMF (1.5 mL), cooled to 0 °C, and DMTMM (7.8 mg, 0.028 mmol) was added. After the addition was complete, the mixture was allowed to return to room temperature and stirred for 2 h. The reaction solution was then purified directly by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% TFA = 5%-50%) to obtain trifluoroacetate of the target compound DL-2 (13.7 mg).

[0247] LCMS (ESI) [M+H] + =913.8.

[0248] 1 H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 2H), 9.10 (s, 2H), 8.97 (d, J =2.4 Hz, 1H), 8.80 (t, J = 6.5 Hz, 1H), 8.12–8.05 (m, 2H), 8.03 (d, J = 7.2Hz, 1H), 8.01–7.94 (m, 2H), 7.87 (d, J = 9.0 Hz, 1H), 7.38 (s, 1H), 5.09 (d,J = 14.8 Hz, 1H), 4.81–4.67 (m, 2H), 4.66–4.55 (m, 1H), 4.30–4.19 (m, 3H), 4.13 (s, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.80 (d, J = 11.2 Hz, 1H), 3.62 (d,J = 14.9 Hz, 2H), 3.40 (s, 3H), 3.26–3.05 (m, 2H), 2.55 (t, J = 7.1 Hz, 2H), 2.30 (t, J = 7.5 Hz, 3H), 2.05–1.87 (m, 2H), 1.86–1.58 (m, 5H), 1.27–1.18 (m,9H).

[0249] Example 3.3: Synthesis of DL-3

[0250]

[0251] Step 1:

[0252] Compound INT5 (28.8 mg, 0.041 mmol) and compound D-1-1 (15.0 mg, 0.041 mmol) were dissolved in DMF (1 mL), cooled to 0 °C, and then DMTMM (12.5 mg, 0.045 mmol) was added. The mixture was stirred at 0 °C for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the formate of the target compound DL-3 (24.0 mg).

[0253] LCMS (ESI) [M+H] + =1048.81.

[0254] 1 H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.10 (s, 2H), 8.97 (s, 1H),8.79 (t, J = 6.5 Hz, 1H), 8.42 (t, J = 5.7 Hz, 1H), 8.23 ​​–8.19 (m, 2H), 8.13(t, J = 5.6 Hz, 1H), 8.04 – 7.93 (m, 3H), 7.84 (d, J = 9.0 Hz, 1H), 7.34 (s,1H), 7.31 – 7.18 (m, 5H), 4.77 (d, J = 6.5 Hz, 2H), 4.53 (td, J = 9.3, 4.5Hz, 1H), 4.36 (d, J = 15.9 Hz, 1H), 4.17 (s, 2H), 4.02 (s, 3H), 3.98 (s, 3H), 3.94 (s, 2H), 3.84 – 3.76 (m, 5H), 3.66 (d, J = 5.5 Hz, 1H), 3.63– 3.58 (m,3H), 3.51 – 3.47 (m, 1H), 3.42 (s, 3H), 3.22 – 3.16 (m, 1H), 3.13 – 3.08 (m,1H), 2.88 – 2.74 (m, 2H), 2.66 (t, J = 7.1 Hz, 2H), 2.36 – 2.30 (m, 1H), 2.22(t, J = 10.7 Hz, 1H), 2.02 (d, J = 10.5 Hz, 1H), 1.92 – 1.72 (m, 4H), 1.63 (d, J = 11.9 Hz, 1H), 1.50 – 1.38 (m, 2H).

[0255] Example 3.4: Synthesis of DL-4

[0256]

[0257] Step 1:

[0258] Compounds INT6 (28.8 mg, 0.041 mmol) and D-1-1 (15 mg, 0.041 mmol) were dissolved in DMF (0.6 mL), cooled to 0 °C, and DMTMM (12.5 mg, 0.045 mmol) was added. After the addition was complete, the mixture was allowed to return to room temperature and stirred for 2 h. The reaction was monitored by LCMS. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 10%-90%) to obtain the target compound DL-4 (19.3 mg).

[0259] LCMS (ESI) [M+H] + =1046.72.

[0260] 1 H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.03 (s, 2H), 8.95 (s, 1H), 8.75 (t, J = 6.5 Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H),8.14 (s, 1H), 8.04 – 7.79 (m, 5H), 7.34 – 7.14 (m, 6H), 4.82 – 4.71 (m, 2H),4.56 – 4.33 (m, 2H), 4.21 – 4.07 (m, 3H), 3.97 (d, J = 16.6 Hz, 6H), 3.88 –3.59 (m, 8H), 3.40 (s, 3H), 3.22 – 3.02 (m, 3H), 2.88 – 2.73 (m, 2H), 2.30 –1.94 (m, 3H), 1.87 – 1.71 (m, 4H), 1.68 – 1.55 (m, 1H), 1.50 – 1.29 (m, 2H), 1.12 (s, 6H).

[0261] Example 5 Synthesis of antibody-drug conjugate (ADC) containing Her2

[0262] Example 5.1: Synthesis of Her2-ADC-001

[0263] Take 3.0 mL of trastuzumab (anti-Her2 antibody, concentration 16.1 mg / mL), dilute with 0.03 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), and adjust the pH to 7.4 with 0.5 M Na2HPO4 solution. Take 1.0 mL of the above solution and add it to 20 mM TCEP (0.0276 mL, 0.552 µmol) solution, mix well, and let stand at room temperature for 90 min. Then add DL-1 (1.257 mg, 12 times the molar amount of antibody) dissolved in dimethyl sulfoxide (0.1205 mL) solution, mix well, and let stand at room temperature for 2 h. After that, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to change the buffer solution to 20 mM His-HCl buffer solution at pH 5.9. The conjugate product Her2-ADC-001 of DL-1 and trastuzumab antibody is obtained. The DAR value determined by mass spectrometry was 8.0 (as shown in Table 5).

[0264]

[0265] Table 5

[0266]

[0267] The DAR value of Her2-ADC-001 was calculated to be 8.0 based on the determination of the light chain conjugation of Trastuzumab antibody with 0-1 toxin linkers (LC, DAR1 ratio 0%, 100%) and the heavy chain conjugation of 0-3 toxin linkers (HC, DAR1, DAR2, DAR3 ratio 0%, 0%, 0%, 100%).

[0268] Example 5.2: Synthesis of Her2-ADC-002

[0269] Using a similar procedure to that in Example 5.1, DL-1 was replaced with DL-2 to obtain the conjugate product Her2-ADC-002 of DL-2 and Trastuzumab antibody. The DAR value was determined by mass spectrometry to be 8.0 (as shown in Table 6).

[0270]

[0271] Table 6

[0272]

[0273] The DAR value of Her2-ADC-002 was calculated to be 8.0 based on the determination of the light chain of Trastuzumab antibody conjugated with 0-1 toxin linkers (LC, DAR1 ratio was 0%, DAR1 ratio was 100%) and the heavy chain conjugated with 0-3 toxin linkers (HC, DAR1 ratio was 0%, DAR2 ratio was 0%, DAR3 ratio was 100%).

[0274] Example 5.3: Synthesis of Her2-ADC-003

[0275] Using a similar procedure to that in Example 5.1, DL-1 was replaced with DL-3 to obtain the conjugate product Her2-ADC-003 of DL-3 and Trastuzumab antibody. The DAR value was determined by mass spectrometry to be 8.0 (as shown in Table 7).

[0276]

[0277] Table 7

[0278]

[0279] The DAR value of Her2-ADC-003 was calculated to be 8.0 based on the determination of the light chain conjugation of Trastuzumab antibody with 0-1 toxin linkers (LC, DAR1 ratio 0%, 100%) and the heavy chain conjugation of 0-3 toxin linkers (HC, DAR1, DAR2, DAR3 ratio 0%, 0%, 0%, 100%).

[0280] Example 5.4: Synthesis of Her2-ADC-004

[0281] Using a similar procedure to that in Example 5.1, DL-1 was replaced with DL-4 to obtain the conjugate product Her2-ADC-004 of DL-4 and Trastuzumab antibody. The DAR value was determined by mass spectrometry to be 8.0 (as shown in Table 8).

[0282]

[0283] Table 8

[0284]

[0285] The DAR value of Her2-ADC-004 was calculated to be 8.0 based on the determination of the light chain conjugation of Trastuzumab antibody with 0-1 toxin linkers (LC, DAR1 ratio 0%, 100%) and the heavy chain conjugation of 0-3 toxin linkers (HC, DAR1, DAR2, DAR3 ratio 0%, 0%, 0%, 100%).

[0286] Example 6: Bioactivity Test of Toxin Molecules

[0287] Example 6.1: Test of the killing activity of toxin molecules against tumor cells

[0288] Experimental Principle and Methods: Gastric cancer cell line N87 and non-small cell lung cancer cell line NCI-H358 were treated with toxin molecules, and their cell proliferation inhibition ability was detected by Cell Titter Glo. Key materials are shown in Table 9.

[0289] Table 9

[0290]

[0291] Experimental Procedure: N87 cells were revived and cultured with 1640+FBS, and seeded at a density of 5000 cells / well in 96-well plates. Incubation was carried out overnight at 37°C. The next day, toxin molecule D-2 (maximum concentration 2 μM) was diluted 4-fold with 1640+FBS for 10 wells. 100 μL of the diluted toxin molecule was added to each well. After incubation at 37°C for 4-7 days, 50 μL of Celltiter glo substrate was added to each well, and fluorescence was detected after 5 minutes of incubation. The half-maximal inhibitory concentration (IC50) of the toxin molecule was determined. 50 (nM) are shown in Table 10.

[0292] Table 10. Detection of the inhibitory activity of toxin molecules on cell proliferation in vitro.

[0293]

[0294] Test results show that the toxin molecules of this invention have a strong killing effect on a variety of tumor cells.

[0295] Example 7 Bioactivity test of ADC

[0296] Example 7.1: Detection of the inhibitory activity of ADC on in vitro cell viability

[0297] Experimental principle and method: Tumor cells were treated with ADC, and their cell proliferation inhibition ability was detected by CellTiter Glo. Key materials are shown in Table 11:

[0298] Table 11

[0299]

[0300] Experimental Procedure: N87 tumor cells and HCC1954 cells were digested using trypsin via standard methods. Cells were collected in tubes and counted. The cells were resuspended in the corresponding detection medium (containing 2% FBS), and 2000-5000 cells / well were added to a 96-well plate. 100 μL of ADC diluted in 2% FBS medium was added to each well of the 96-well plate, starting at a concentration of 150 μg / ml and then 3-fold diluted (12 concentration gradients). The plates were incubated at 37℃ with 5% CO2 for 4-7 days. Then, 20 μL of CCK8 reagent was added to each well, and the reaction was allowed to proceed for 2-6 hours. The readings were taken using a microplate reader (detection wavelength 450 nm), and the half-maximal inhibitory concentration (IC50) was calculated. 50 (nM), the results are shown in Table 12.

[0301] Table 12. Detection of the inhibitory activity of ADC on cell proliferation in vitro.

[0302]

[0303] In the table, " / " indicates that it was not detected.

[0304] Test results show that the ADC molecule of the present invention has a strong killing effect on a variety of tumor cells.

[0305] Example 7.2: Efficacy test of ADC on N87 xenograft tumors

[0306] 1. Experimental Materials

[0307] Subjects: Herceptin, Her2-ADC-001, and saline as a negative control.

[0308] Experimental cells: NCI-N87 cells.

[0309] Experimental animals: Balb / c Nude mice, female, 5-6 weeks old, purchased from Chengdu Yaokang Biotechnology Co., Ltd.

[0310] 2. Experimental Design

[0311] 2.1. Cell Treatment

[0312] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in culture flasks. When the cell confluence reached approximately 80-90%, they were digested with trypsin containing EDTA. The cells were washed twice with PBS, then centrifuged and resuspended in pre-chilled PBS. Cells were counted using a cell counter, and the cell suspension was adjusted to an appropriate concentration before seeding.

[0313] 2.2. Tumor cell transplantation

[0314] Balb / c Nude mice were acclimatized to the laboratory environment for 3-5 days, and then NCI-N87 cells were subcutaneously injected into the right rib area at a cell density of 5 × 10⁶ cells. 6 / animal, inoculated with 0.2 mL (containing 50% Matrigel), until the tumor grows to 200 mm 3 When the time is around 10:00, administer the medication in groups.

[0315] 2.3. Animal drug administration and detection

[0316] The tumor-bearing nude mice enrolled in the group were administered medication according to the regimen shown in Table 13:

[0317] Table 13 Dosing Regimen

[0318]

[0319] 2.4. Tumor volume and body weight measurement

[0320] Tumor volume and body weight were measured twice a week, and the relative tumor proliferation rate (T / C) and relative tumor inhibition rate (TGI) were calculated.

[0321] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 ;

[0322] Relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100% (TRTV: mean RTV in the treatment group; CRTV: mean RTV in the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment).

[0323] Relative tumor inhibition rate (TGI) (%) = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively).

[0324] 3. Experimental Results

[0325] The test results showed that all the ADC drugs disclosed in this paper exhibited significant tumor-suppressing effects. During the administration period, no significant weight loss or drug toxicity was observed in any of the groups of animals. Specific results are shown in Table 14.

[0326] Table 14 Tumor volume data of NCI-N87 xenograft model

[0327]

[0328] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody-drug conjugate and one or more pharmaceutical excipients; the antibody-drug conjugate is: , where q is 8.

2. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate has the following structure: , where q is 8.

3. A drug linker conjugate, characterized in that, The drug linker conjugate has the following structure: 。 4. Use of the antibody-drug conjugate according to claim 2 or the pharmaceutical composition according to claim 1 in the preparation of medicaments for treating and / or preventing diseases related to abnormal cell activity; wherein, The diseases associated with abnormal cell activity are selected from cancer, and the cancer is selected from gastric cancer.

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